1. Field of the Invention
The present invention relates generally to the field of transgenic animals. More particularly, it concerns methods for generating primordial germ cell-derived cell lines, transforming primordial germ cells and primordial germ cell-derived cell lines, and using these transformed cells and cell lines to generate transgenic non-rodent animal species.
2. Description of Related Art
Animals having certain desired traits or characteristics, such as increased weight gain, feed efficiency, carcass composition, milk production or content and disease resistance have long been desired. Traditional breeding processes are capable of producing animals with some desired traits, but these are often accompanied by a number of undesired characteristics, and is an extremely costly and time consuming process.
The development of transgenic animal technology holds great promise for the production of animals having specific, desired traits. Transgenic animals are animals that carry a gene that has been deliberately introduced into somatic and germline cells at an early stage of development. Although transgenic animals have been produced by various methods in several different species, methods to readily and reproducibly produce transgenic large mammals at reasonable costs are still lacking.
At present the only techniques available for the generation of transgenic domestic animals are by pronuclear injection or use of viral vectors. In both cases the incoming DNA inserts at random, which can cause a variety of problems. The first of these problems is insertional inactivation, which is inactivation of an essential gene due to disruption of the coding or regulatory sequences by the incoming DNA. Another problem is that the transgene may either be not incorporated at all, or incorporated but not expressed. A further problem is the possibility of inaccurate regulation due to positional effects. This refers to the variability in the level of gene expression and the accuracy of gene regulation between different founder animals produced with the same transgenic constructs. Thus, it is not uncommon to generate 10 founder animals and identify only one that expresses the transgene in a manner that warrants the maintenance of the transgenic line.
Additionally, using the present technology, it is not possible to fully inactivate or remove genes in transgenic animals, only add new genes. As a result it is not possible to delete genes involved in undesired cellular processes, or to undertake any genetic modification that entails changes in existing genes. Moreover, the efficiency of generating transgenic domestic animals is low, with efficiencies of 1 in 100 offspring generated being transgenic not uncommon (Wall, 1996). As a result the cost associated with generation of transgenic animals can be as much as 250-500 thousand dollars per expressing animal (Wall, 1996).
These drawbacks are overcome by the utilization of homologous recombination (Koller and Smithies, 1992), which directs the insertion of the transgene to a specific location. This technique allows the precise modification of existing genes, and overcomes the problems of positional effects and insertional inactivation. Additionally, it allows the inactivation of specific genes as well as the replacement of one gene for another. Unfortunately the efficiency of the procedure is so low that it cannot be utilized directly on embryos but must make use of a carrier cell line. The availability of appropriate cell lines will allow the precise manipulation of the genomic material followed by the generation of a living animal carrying those changes.
Embryonic stem (ES) cells, isolated from the inner cell mass (ICM) of the preimplantation embryo, possess the ability to proliferate indefinitely in an undifferentiated state, and are capable of contributing to the formation of normal tissues and organs of a chimeric individual when injected into a host embryo. The ES cell line allows manipulation and selection in vitro, followed by the generation of a transgenic animal carrying those changes. The ability to colonize the germ line following culture and genetic manipulation have made ES cells a powerful tool for the modification of the genome in the mouse species. Chimeras produced between genetically modified ES cells and normal embryos have been used to study in vivo gene regulation (Stewart et al., 1985), as well as germ-line transmission of introduced genes (Smithies 1991). In addition, ES cells have been used to study targeted modification of genes by homologous recombination (Smithies 1991).
The use of chimeras has been shown to be effective in producing transgenic mice. About 70% of expanded mouse blastocysts develop into live young with about 50% of the young born being chimeric (Bradley et al., 1984). Twenty percent of these chimeric young have germ cell chimerism. Utilizing this method it is possible that chimerism in the germ line may be 20-30%. However, the ES-cell method has not been successfully applied to production of larger transgenic mammals, for example, transgenic pigs, cattle, goats or sheep. A reason for the failure to extrapolate methods from mice to larger mammals may be the difference in developmental stages of the species (Wheeler, 1996).
Recently, it has been reported that murine cell lines derived from primordial germ cells (PGC) behave similarly to ES cells and are capable of contributing to the germ line (Labosky et al., 1994). These cells, referred to as embryonic germ (EG) cells or PGC-derived cells (Labosky et al., 1994; Strelchenko, 1996), are indistinguishable from ES cells in terms of markers of the undifferentiated state as well as their ability to colonize the germ line following injection into host blastocysts (Labosky et al., 1994; Stewart et al., 1994). Thus, even though the starting tissue source or cellular phenotype differ from the ICM-derived cell lines, once established they have similar, if not identical, properties.
Although the majority of the research on ES and primordial germ cells has been done in the mouse, attempts at developing this technology in other mammalian species have been reported. Embryonic cell lines have been described from hamster (Doetschman et al. 1988), mink (Sukoyan et al., 1992, 1993), rabbit (Graves and Moreadith, 1993; Giles et al., 1993), pig (Piedrahita et al., 1990; Strojek et al., 1990; Notarianni et al., 1990; Talbot et al., 1993; Wheeler, 1994; Gerfen and Wheeler, 1995; Shim and Anderson, 1995), sheep (Handyside et al., 1987; Piedrahita et al., 1990; Notarianni et al., 1991; Campbell et al., 1995) and cattle (Saito et al., 1992; Sims and First, 1993; Stice et al., 1994; Strelchenko, 1996; Stice and Strelchenko, 1996). Although each of these cell lines have some of the characteristics of the ES cells described from mice, germ line transmission, a prerequisite for generation of a transgenic line of animals, has not been demonstrated.
Another problem associated with the generation of transgenic animals is the difficulty with transformation of ES or EG cells with DNA carrying a desired trait or traits. These difficulties are related to the inability of the cells to remain unchanged (undifferentiated) upon repeated passage. This is in contrast with mouse ES cells, which can be passaged multiple times without major changes in the potential to generate a transgenic animal. To date there have been no reports on the generation of undifferentiated transformed transgenic cell lines of embryo-derived or PGC-derived cell lines in any non-rodent domestic animal species.
A genetically transformed ES or PGC-derived cell line capable of taking part in chimera formation, or nuclear transfer development in enucleated oocytes, would be of great value for the medical, veterinary, and agricultural community. In the medical and veterinary field it would allow the generation of biopharmaceuticals and oral immunogens in the milk, the generation of animals that can be used as human tissue donors, the development of animal models of human disease that can speed the development of alternative therapeutic methods including gene therapy, and the development of blood substitutes. In the veterinary field it would allow the generation of animals that are naturally immune to particular diseases. In the agricultural field it will allow the modification of the milk composition to increase shelf life, cheese yield, and permit lactose intolerant individuals to safely consume the modified milk. It will also allow the introduction of small genetic changes that can modify disease resistance, growth rate and carcass composition, wool composition, and nutritional efficiency, among others. Unfortunately, to date, there has been no description of transformed non-rodent ES or PGC-derived cell lines.